In October 2013, a new disease affecting purple woodnettle, Oreocnide pedunculata, plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'-broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of polymerase chain reaction with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV-rpsC region revealed that purple woodnettle witches'-broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI-AH and rpI-Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI-B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV-rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris-related strain in 16SrI-AH, rpI-Q and tufI-B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.
Carrot (Daucus carota) is an important root vegetable planted and consumed worldwide (Stein and Nothnagel 1995). In June 2020, carrots (cv. New Kuroda) showing soft rot symptoms were observed in a 600 sqft plot located in Pitou, Changhua, Taiwan (23°54'00.9"N, 120°28'37.3"E; with around 400 plants). About 10% of the plants on site had similar symptoms; infected taproot tissues were macerated (Figure S1) and emitted a foul odor. In most cases, the peels above the rotten tissues remain intact. Two infected plants were brought to the lab. Macerated tissues were suspended in water and examined under a microscope at 600X (without staining). Rod, motile bacteria were observed in all of the samples and the bacteria were isolated onto nutrient agar. Three bacterial strains were obtained from two taproots; strain Car1 was isolated from one plant, and strains Car2 and Car3 were isolated from the other. Their colonies were translucent, round and convex. All isolates could ferment glucose and induce soft rot symptoms on potato tuber slices (Schaad et al. 2001). They were not able to produce indigoidine on yeast dextrose calcium carbonate agar and were tested negative for phosphatase activity (Schaad et al. 2001). The 16S rDNA of Car1 to Car3 were amplified using primers 27F/1492R (Lane 1991). Cloning and sequencing of their 16S rDNA (GenBank accession no. MT889640) revealed that their sequences shared 99.9% identity (1,463/1,464 bp) with that of Pectobacterium aroidearum CFBP 8168T (SCRI 109T; GenBank accession no. NR_159926.1). Multilocus sequence analyses targeting the three isolates’ dnaX, leuS and recA genes were conducted. The concatenated sequences (1,596 bp) of Car1 to Car3 and those included in a previous work (Portier et al. 2019) were subjected to phylogenetic analysis. The sequences of Car1 to Car3 were identical (GenBank accession nos. MT892671-MT892673). A maximum-likelihood tree showed that the three isolates belonged to the same clade as P. aroidearum CFBP 8168T (GenBank accession nos. MK516971, MK517115 and MK517259; Figure S2). For the concatenated sequences analyzed, the identity between P. aroidearum CFBP 8168T and our three isolates was 99.4% (1,587/1,596 bp). The pathogenicity of these isolates was determined by inoculating the bacteria into carrot (cv. Xiangyang No.2) taproots. Strains Car1 to Car3 were grown on NA for 48 h (28 °C) and cell suspensions with OD600 values of 0.3 (2.4 x 108 CFU/ml; in water) were prepared. The suspensions of each strain (100 μl) were loaded into 200 μl pipette tips. The tips were then pierced into intact carrot taproots (2.4 cm deep), ejected and left on the plants (one tip per plant). Three taproots were tested for each strain. Tips loaded with 100 μl of water were used for the controls (three replicates). The plants were incubated in a sealed plastic container kept in a growth chamber set at 28°C. After 48 h, all of the inoculated taproots produced soft rot symptoms resembling those observed in the field and plants in the control group did not. Bacteria were re-isolated from macerated tissues of the artificially infected plants and found to share the same leuS sequence with Car1 to Car3. Occurrences of carrot soft rot in Taiwan have only been attributed to Dickeya spp. (Erwinia chrysanthemi) in previous studies (Hsu and Tzeng 1981). The present study is the first report of P. aroidearum infecting carrots in Taiwan. The findings may add to our understanding of the diversity of soft rot pathogens affecting carrot production in Taiwan.
Scindapsus pictus (satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage ornamental (Masnira et al. 2019). In September of 2022, soft rot symptoms were observed on potted S. pictus plants grown in a greenhouse in Nantun District, Taichung, Taiwan, in which soft rot of another aroid (philodendron) has also been reported (Wu et al. 2023). The symptoms appeared on the petioles and most of them tended to extend to the leaf blades; the colors of leaf lesions ranged from dark brown to gray (Fig. S1). Some 70% of the plants in the greenhouse showed similar symptoms and losses were estimated to be 15-30%. Four symptomatic plants were sampled. Macerated tissues from rotting petioles were soaked in 10 mM MgCl2 and observed under a light microscope (Nikon, Japan) at 400 x magnification. Motile, rod-shaped bacteria were observed, and 1-2 loopfuls of undiluted sample suspension were streaked onto nutrient agar (NA; Gibco, USA). After culturing at 28°C for 1 day, all samples yielded round, creamy-white colonies (0.9 mm in diameter) and from each of the four samples a pure culture was obtained (Spi1-Spi4). All isolates exhibited oxidative and fermentative metabolism of glucose (Schaad et al. 2001). They caused pitting on crystal violet pectate agar, induced maceration on potato tuber and were tested positive for phosphatase activity and indigoidine production (Lee and Yu 2006; Schaad et al. 2001). Polymerase chain reaction tests using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon (0.5 kb) in extracted DNA samples of all isolates. Identification of the strains was achieved by amplifying and sequencing fragments of the housekeeping genes gyrB, recN, dnaA, dnaJ, and dnaX (Marrero et al. 2013); the lengths of the five gene fragments analyzed were 822, 762, 720, 672, and 450 bp, respectively (accession nos. OP985528-OP985532). The five sequences were concatenated for every isolate; the resulting 3,426 bp sequences were aligned with ClustalW and found to be identical. A maximum-likelihood analysis was conducted using the 3,426-bp sequences and those of known Dickeya species’ type strains. Spi1 to Spi4 clustered with D. dadantii subsp. dieffenbachiae NCPPB 2976T and D. dadantii subsp. dadantii CFBP 1269T (Fig. S2) with sequence identities of 98.4 and 98%, respectively. To fulfil Koch’s Postulates, stab inoculations of the four isolates into the petioles of cutting propagated, 38-day-old S. pictus plants (3 plants per isolate) were conducted using sterile toothpicks. The amounts of bacteria used was approximately 106 cfu per toothpick; the bacterial loads were estimated by suspending the cells in 10 mM MgCl2 and spread-plating diluted suspensions on NA. Sterile toothpicks were used as control. All tested plants were sealed in plastic bags (containing wet paper towel) and kept in a growth chamber (28°C; 12-h photoperiod). After 1 day, all isolates induced soft rot symptoms resembling those observed under natural conditions in the greenhouse. Bacteria were re-isolated, and they all shared the same dnaX sequence with strains Spi1 to Spi4. This is the first report of S. pictus affected by D. dadantii in Taiwan. Further investigation is needed to determine whether Spi1-Spi4 belong to D. dadantii subsp. dieffenbachiae. Dickeya dadantii has been found infecting different aroids (Lee and Chen 2021; Lin et al. 2012). The species has also been reported in Taiwan on poinsettia (Wei et al., 2019) and philodendron (Wu et al. 2023). Because these plants are often grown closely in the same facilities, growers should be wary of D. dadantii’s spread among these plants. Reduction of environmental humidity and avoiding overhead irrigation may be effective in preventing the pathogen’s transmission.
The sweet William (Dianthus barbatus) is an ornamental belonging to the Caryophyllaceae family; the species produces clusters of flowers that comes in various colors and is grown commonly as garden plants (Lim 2014). In February 2021, sweet Williams showing symptoms typical of phytoplasma diseases were found in a garden located in Wufeng District, Taichung, Taiwan (24°04'37.6"N 120°43'20.4"E). Infected plants exhibited virescence and phyllody symptoms and produced an abnormal number of new shoots from the base of the flowers/flower-like structures (Figure S1) as well as the base of the plants. Among the fifteen plants grown in the area, two exhibited such symptoms. The two symptomatic plants, along with five symptomless plants were sampled. Two flower-like structures were collected from each of the symptomatic plants, and two flower samples were collected for each symptomless plant (Figure S2). Total DNA were extracted from each sample using the Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) and subjected to diagnostic PCR using primers P1/P7 (Schneider et al. 1995). All four symptomatic samples produced a 1.8-kb amplicon and the ten symptomless samples did not. The amplification products were diluted fifty-fold and used in a second round of PCR using primers R16F2n/R16R2 (Gundersen and Lee 1996). Again, only the symptomatic samples produced an expected 1.25-kb amplicon. A sample was selected for each plant and the PCR products from the first round of PCR were cloned using the pGEM-T Easy Vector System (Promega Inc.) and sequenced (three clones per sample). Fragments of the 16S rRNA gene (1,248 bp; GenBank accession: MW788688) were analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). Sequences obtained from the two infected plants were identical, and were classified to the 16SrII-V subgroup with similarity coefficients of 1.0; they also shared 98.6% similarity with the sequence of a 'Candidatus Phytoplasma aurantifolia' reference strain (accession: U15442). BLASTn results indicated that the 16S rRNA gene sequences detected were identical to those of 16SrII-V phytoplasmas affecting mungbean (accession: MW319764), lilac tasselflower (accession: MT420682), peanut (accession: JX403944) and green manure soybean (accession: MW393690) found in Taiwan. To corroborate the above results, 16SrII group-specific primers were used to conduct nested and semi-nested PCR targeting the pathogen’s 16S rRNA gene (outer primers: rpF1C/rp(I)R1A; inner primers: rp(II)F1/rp(II)R1; Martini et al. 2007) and immunodominant membrane protein gene (imp; outer primers: IMP-II-F1/IMP-II-R1; inner primers: IMP-II-F2/IMP-II-R1; Al-Subhi et al. 2017). In both assays, the symptomatic samples produced the expected amplicons and the symptomless samples did not. The coding sequence of the imp gene (519 bp; accession: MW755353) was the same among all symptomatic samples, and shared 100% identity with that of the peanut witches'-broom phytoplasma (16SrII; accession: GU214176). To our knowledge, this is the first report of a 16SrII-V phytoplasma infecting sweet Williams in Taiwan. Since 16SrII-V phytoplasmas have also been found infecting mungbeans and peanuts in Taiwan (Liu et al. 2015), the findings here suggest that by serving as a natural host in the field, the sweet William may potentially contribute to the spread of 16SrII-V phytoplasmas to food crops.
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